PROJECT SUMMARY/ABSTRACT Enterococcus faecalis significantly contributes to the burden of escalating healthcare costs as a leading cause of healthcare-associated infections. The antimicrobial-recalcitrant nature of E. faecalis and its ability to form biofilms necessitates prolonged and complex treatment strategies for infections. Therefore, there is a critical need to identify new approaches for treating and preventing enterococcal disease. We previously showed that the conserved intramembrane metalloprotease Eep, part of the site 2 protease (S2P) family, is a critical factor in E. faecalis for both in vitro biofilm formation and in vivo biofilm-associated infection. This largely uncharacterized role of Eep in E. faecalis biofilm formation is in addition to its documented functions in cell-cell signaling, cellular adaptation in response to attack by the innate immune system effector molecule lysozyme, and the spread of antibiotic resistance-carrying plasmids. However, despite its importance in these cellular processes, there are numerous unanswered fundamental questions about Eep?s biochemical activity and how that activity influences E. faecalis pathogen-host interactions. More broadly, our mechanistic and biological understanding of Eep and orthologous S2Ps in other Gram-positive pathogens is also limited. This project will investigate the following questions: (1) What are Eep?s substrates and products?; (2) How do Eep?s effectors affect biofilm formation and adaptation to cell surface stress? In turn, how do these Eep-dependent processes affect host-pathogen interactions?; and (3) How does Eep?s structure influence its ability to recognize and cleave substrates? Our experimental design will test the hypothesis that the proteolytic activity of Eep leads to coordinated changes at the cell surface that influence E. faecalis interactions with mammalian hosts in pathogenic settings. We will pair proteomic, molecular genetic, and microbiological approaches with two animal models of in vivo biofilm formation to identify candidate Eep substrates and downstream effectors in biofilms (Aim 1), characterize the genetic and biochemical basis of cell surface alterations that render E. faecalis cells resistant to lysozyme (Aim 2), and determine the key structural regions and amino acids in Eep that contribute to its function (Aim 3). Completion of the proposed experiments will provide fundamental new knowledge about the functions and mechanism of a conserved enzyme that will be translatable to other pathogenic bacteria that are associated with antibiotic resistance and biofilm infections, such as methicillin-resistant Staphylococcus aureus and Clostridium difficile.